Technical Field
The invention relates to transgenic animals with progressive neurologic disease characterized by both behavioral and neuropathological changes as compared to nontransgenic age-matched animals and their use for screening for agents which can be used to treat or cure progressive neurologic syndromes such as Alzheimer""s disease. The invention is exemplified by transgenic mice which express native or mutant xcex2-amyloid precursor protein in brain tissue at superendogenous levels under control of prion protein gene regulatory sequences.
Background
The term degenerative as applied to diseases of the nervous system is used to designate a group of disorders in which there is gradual, generally relentlessly progressing wasting away of structural elements of the nervous system; many of the conditions so designated depend upon abnormal genetic factors. The degenerative diseases manifest themselves by a number of syndromes distinguished by their clinical and pathological features. Nevertheless, there are certain aspects common to all. These aspects include a gradually progressive course of disease onset, bilaterally symmetric distribution of the changes brought about by the disease, and in many cases, the almost selective involvement of anatomically or physiologically related systems of neurons. Typically the pathologic process is one of slow involution of nerve cell bodies or their prolongations as nerve-fibers.
Among the degenerative diseases of the nervous system are syndromes in which the outstanding feature is progressive dementia; the syndromes in this group include senile dementia and Alzheimer""s disease. Senile dementia is a fairly frequent condition of old age, not only in humans but also in other animals. Alzheimer""s disease is a pathologically identical, but much more infrequent, progressive dementia which come son well before the senile period. The distinction between the two conditions is purely clinical; pathologically they differ only in that the characteristic abnormalities tend to be more severe and widespread in cases of Alzheimer""s disease and to begin at an earlier age than at the senile period.
Alzheimer""s disease (AD) shows a slowly progressive mental deterioration with failure of memory, disorientation and confusion leading to profound dementia. The disease predominantly involves limbic and cortical regions of the brain. There are several histologic features, but two are striking. First, argyrophilic plaques containing the amyloidogenic Axcex2 fragment of amyloid precursor protein (APP) are scattered throughout the cerebral cortex and hippocampus. Second, neurofibrillary tangles are found in pyramidal neurons predominantly located in the neocortex, hippocampus, and nucleus basalis of Meynert. There are other changes, also. Granulovacuolar degeneration in the pyramidal cells of the hippocampus, which have been considered by some to be more specific for AD than plaques or neurofibrillary tangles, are observed. Finally, there is neuronal loss and gliosis in the cortex and hippocampus.
There are patients with dementia who lack the pathologic features of AD (and therefore by definition have a different disease), and conversely, there are individuals with many of the pathologic features of AD who were not demented prior to death. A diagnosis of AD requires that both the clinical and the pathological features characteristic for the disease be present in the patient; the diagnosis cannot be made with certainty from either clinical or pathological features alone. Whether neural dynsfunction and clinical abnormalities precede the development of the pathologic features, particularly the amyloid plaques and neurofibrillary tangles, is unknown.
The clinical manifestations of AD predict the regions of affected brain structures in the forebrain, including the cerebral cortex, hippocampus, amygdala, and parahippocampal gyri. These regions are known as the cortico-limbic areas of the brain. The hindbrain is spared, including the cerebellum, the pontine and the medullary nuclei. Within the cerebral neocortex, the primary cortical area is relatively spared, which corresponds to the relative sparing of basic motor and sensory cortical functions observed clinically.
Research into progressive neurologic disorders such as AD, and means for screening for agents which can be used to treat or cure these disorders, has been seriously impeded by the lack of easily accessible animal models. Some aspects of the neuropathology of aged primates are similar to those of human AD (Price, et al., (1992) J. Neurobiol, 23:1277-1294). Aged primates develop amyloid plaques and forme fruste neurofibrillary tangles. No other animals studied develop a disease resembling AD as closely as do aged primates; aged primates are impractical to study in large numbers and their use raises both moral and economic issues.
Transgenic mice harboring APP transgenes have been described; however, the reported transgene product expression falls considerably short of endogenous levels of APP; total APP levels in these order transgenic mice have not exceeded 150% of endogenous levels, and fails to generate a disease phenotype with a progressive neurobehavioral disorder accompanied by pathology in the cortico-limbic regions of the brain. In these other transgenic mice, there have been no signs of progressive neurologic disorder or of neuropathologic changes in the brain which may be regarded as evidence of a true neurologic disease nor have changes such as neurobehavioral changes which can be used in live animals as a means of screening for agents which prevent, ameliorate or cure a progressive neurologic disorder been described.
Missense point mutations in the gene coding for amyloid precursor proteins have been linked to familial AD. However, despite the discovery of disease associated mutations in APP, most published attempts to create transgenic animals with AD have involved only wild-type APP transgenes in mice (Kawabata, et al., (1991) Nature 354, 476-478; Quon, et al., (1991) Nature 352, 239-41; Wirak, et al., (1991) Science 253, 323-325; Kammesheidt, et al., (1992) Proc Natl Acad Sci U.S.A. 89, 10857-61; Lamb, et al., (1993) Nature Genetics 5, 22-30.) Unfortunately, several of the published studies purporting pathology have been confounded by inadequate documentation of transgene product expression and/or misinterpretation of pathology. Two have been retracted (Kawabata, et al., (1991) Nature 354, 476-478; and Wirak, et al., (1991) Science 253, 323-325.
Previous efforts to create a model of AD in transgenic mice have been discouraging. In most cases, transgene product expression comparable to or exceeding endogenous levels of APP was not achieved and the transgenes did not encode mutated APP. PCT/US92/11276 reports methods for using mutant genes. In some cases, the entire APP gene was not expressed, just the carboxyl terminus (Kammesheidt, et al., (1992) Proc Natl Acad Sci U.S.A. 89, 10857-61); expression of only the carboxyl terminus of APP may overlook any biologic effect that the rest of the APP molecule may exert in AD.
Preamyloid APP plaques have been observed in some transgenic mice. However, preamyloid APP plaques are not necessarily indicative of a disease, since they are routinely observed in human brain regions, such as the cerebellum, which are devoid of other signs of pathology or clinical manifestations. Increased APP immunoreactivity located within vesicular structures in hippocampal neurons of transgenic mice has been reported, but the significance of this immunoreactivity is unclear since the mice exhibited neither a progressive neurobehavioral disorder nor evidence of true neuropathology.
In general, the ceaselessly progressive course of neurodegenerative diseases is uninfluenced by current treatment modalities. It therefore is of interest to develop a transgenic non-human animal model for degenerative neurologic diseases such as senile dementia and AD wherein the animal develops a progressive degenerative neurologic disease of the cortico-limbic brain resembling the disease, both clinically and pathologically (e.g. the gliosis and the specific brain regions affected). It also is desirable that the animal develops neurologic disease within a fairly short period of time from birth, facilitating the analysis of multigenerational pedigrees. The model can be used to study the pathogenesis and treatment of degenerative neurologic diseases since there is a distinct and robust clinical and pathologic phenotype to examine and score in the live animal.
Transgenic mice (Swiss Webster x C57B6/DBA2 F1) expressing three isoforms of mutant xcex2APPV717F with an overrepresentation of KPI-containing isoforms show Alzheimer-type neuropathology including abundant thioflavin S-positive Axcex2deposits, neuritic plaques, synaptic loss, astrocytosis and microgliosis (Games, et al., Nature 373:523-527 (1995)), but deficits in memory and learning have not yet been reported. Transgenic mice (JU) expressing human wild-type xcex2APP751 show deficits in spatial reference and alternation tasks by 12 months of age (Moran, et al., Proc. Natl. Acad. Sci. USA 92:5341-5345 (1995)) but only 4% of aged ( greater than  greater than 12 months) transgenic mice exhibited rare diffuse Axcex2 deposits that do not stain with Congo red dye (Higgins, et al., Annals of Neurology 35:598-607 (1994)). Quon, et al. (1991) Nature 352:239 describe transgenic mice containing human amyloid precursor protein genes. Lamb, et al. (1993) Nature Genetics 5:22 describe transgenic mice in which the amount of amyloid precursor protein expressed is approximately 50% over endogenous levels. PCT application US92/11276 discloses methods for constructing transgenic mice and rats which would express, under various promoters, three forms of the xcex2-amyloid precursor protein (APP), APP695, APP751, and APP770. No data are provided in the specification as to whether APP expression is obtained in vivo using these methods. Also see U.S. Pat. No. 5,455,169 and WO 9213069.
Other transgenic mouse studies of Alzheimer amyloid precursor (APP) protein expression include the following. Greenberg, (1993) Abstract 421.12, Society for Neuroscience Abstracts 19:1035 discloses APP protein gene expression using MAPP and mMt-I promoters. Schwartz, et al. ((1993) Abstract 421.13, Society for Neuroscience Abstracts, 19:1035) disclose neuron-specific expression of human xcex2-amyloid precursor protein (APP) in transgenic mice. Savage, et al. ((1993) Abstract 421.14 Society of Neuroscience Abstracts 19:1035) disclose human amyloid precursor protein expression in transgenic mice as a model of Alzheimer""s disease. Lieberburg, ((1993) Abstract 421.15, Science for Neuroscience Abstracts 19:1035) disclose expression of human amyloid precursor protein in transgenic mice using the NSE promoter. Fukuchi, et al. ((1993) Abstract 421.16, Society for Neuroscience Abstracts 19:1035) disclose intestinal xcex2-amylolidosis in transgenic mice. A chicken xcex2-actin promoter and CMV enhancer were used for expressing the APP protein gene.
Wagner, et al. ((1983) Proc. Natl. Acad. Sci. U.S.A. 78:5016) describe transgenic mice containing human globin genes. Scott, et al. ((1989) Cell 59:847) describe transgenic mice containing hamster prion protein genes. Hsiao, et al. ((1990) Science 250:1587) describe transgenic mice containing mutant human prion protein genes. Hsiao disclosed a model for Gerstmann-Straussler-Scheinker disease (GSS), a rare neurodegenerative disease caused by mutations in the prion protein (PrP) gene, in transgenic mice in which levels of mutant transgene product exceeding endogenous levels were needed to generate a clinical and pathological phenotype (Hsiao, et al. (1990) Science 250:1587-1590); Hsiao, et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:9126-9130).
A transgenic non-human animal model for progressive neurologic disease is provided, together with methods and compositions for preparation of the animal model and methods for using it. The non-human mammals are obtained by the steps of introducing multiple copies of an expression cassette into the non-human mammal at an embryonic stage, and developing the embryo to term in a pseudo-pregnant foster female. The expression cassette comprises an amyloid precursor protein coding sequence operably joined to regulatory sequences for expression of the coding sequence in neurologic tissues at a level at least two to four-fold that of endogenous levels of wild-type amyloid precursor protein. The resulting transgenic non-human mammals develop progressive neurologic disease in the cortico-limbic areas of the brain. The transgenic animals find use for example in screening protocols for agents which can be used for treatment and/or prevention of progressive neurologic diseases.